Cell-penetrating peptides (CPPs) are short peptides able to cross the cellular membranes without any interaction with specific receptors. Thanks to their ability to transport various cargo inside the cells are emerged as powerful therapeutic agents alternative to small molecules. In recent years, numerous preclinical studies provided promising results for the treatment of various human diseases. Several CPP-conjugated compounds are under clinical trials.

In a recent issue of this journal Eissa et al. highlight a novel membrane-active peptide, named EJP18, which is derived from the juxtamembrane domain of epidermal growth factor (EGF) receptor. The authors have characterized the L- and D- form of this peptide and showed that both enantiomers are able to penetrate into cells and to deliver small and large cargoes. This work offers a novel active peptide on the cell-penetrating peptides’ (CPPs) market today. Although a comprehensive understanding of intrinsic properties and intermolecular interactions are required, EJP18 could be potentially suitable for cancer therapies.

Besides some limitations remain to be overcome, the use of CPPs and CPP-conjugates is increasingly feasible and therefore they deserve continued investigation.

Over the last decades, membrane-active peptides are emerged as important therapeutic agents alternative to small molecules. Among them, CPPs, also known as protein transduction domains, are a class of diverse short peptides, typically with 5–30 amino acids, that can cross the cellular membrane [1,2].

CPPs have received more attention when it has been unravelled that beyond owing ability to deliver themselves, they are able to deliver a wide variety of cargoes into cells. So far, small drugs, nucleic acids (siRNAs, antisense oligonucleotides, DNA), other peptides and large proteins have been conjugated, by either covalent or non-covalent bonds, to CPPs and penetrate successfully within cells. Let us say, CPPs are now considered peptidic delivery shuttles into the cells.

Since their discovery in 1988 [3,4], more than 1700 natural (protein-derived) and synthetic CPPs have been developed and validated in vitro and in vivo model systems. In the recent years, preclinical studies provided promising results for the treatment of various human diseases [5,6]. Interestingly, some CPP-conjugates are in phase II or III clinical trials for different applications such as inflammation, cardiovascular diseases, muscular dystrophy, cancer [5,6].

Based on their physicochemical properties, CPPs can be classified into three classes: cationic, which comprises peptides with net positive charges, mainly arginines; amphipathic, containing both polar and nonpolar amino acids that in some cases can assume α-helical structure; hydrophobic, the less abundant group which encompasses peptides with apolar residues and with low net charge [1,2]. The chemical and structural diversity of peptide sequences results in the different mechanisms of uptake as well as in the different rate of uptake. Although not fully clarified, direct penetration and endocytosis are the two main pathways through which CPPs enter cells [1,2,5]. Except for hydrophobic peptides, which seem naturally translocate across membranes, endocytosis is the major mechanism used by CPPs, at least at lower concentrations. Of note, different endocytic routes, such as macropinocytosis, clathrin-dependent and caveoale and/or lipid raft-dependent endocytosis, have all been involved in CPP entry [7]. Importantly, the route undertaken by CPPs and their conjugates is crucial for their bioavailability and activity.

In this issue, Eissa et al. [8] reported the identification of a novel membrane-active peptide, named EJP18 (EGFR Juxtamembrane Peptide-18), which is derived from the juxtamembrane domain of EGF receptor. In this study, the authors have investigated the properties of L- and D- form of this peptide in terms of cytotoxicity, cellular uptake and cargo delivery using four cancer cell lines (cervix adenocarcinoma HeLa, breast adenocarcinoma MDA-MB-231, epidermoid carcinoma A431, acute myeloid leukaemia KG1a).

Interestingly, Eissa et al. [8] reported that EJP18 has a different impact at diverse concentrations. Time-lapse experiments show that all cell lines exhibit large plasma membrane protrusions and extensive blebbing at high concentrations of both enantiomers (above 10 μM). Instead, the plasma membrane integrity is preserved at lower concentrations [8]. Many studies have reported similar killing effects for some CPPs, such as MAP and transportan, on various cells in a dose-dependent manner [2,9]. All together, these findings point out that peptide concentration is a critical parameter for CPP action. CPPs might act as double edge sword mediating different biological effects in dependence on their concentrations (Figure 1). Whether CPPs might elicit their cytotoxic effects on surface and/or organelle membranes or through interactions with specific cellular components remains to be elucidated.

At lower concentrations (1–2 μM) both fluorophore-conjugated enantiomers of EJP18 were detected in intracellular punctate structures throughout the cytoplasm both in HeLa and MDA-MB-231 cells, indicating that its uptake is primarily endocytic. Appreciable fluorescence was also revealed in both cell lines after incubation with eGFP- or fluorescent BSA-EJP18 conjugates, showing its ability for promoting cargo delivery. It is worth noting that both fluorescent EJP18 and eGFP-EJP18 appear to traffic differently in the two cell lines, inferring that CPPs use different pathways in dependence on the cell type and on the size and physical–chemical properties of attached cargoes as previously shown [10,11]. The comprehension of the fate of this peptide as, more generally, of CPPs is fundamental for understanding their mechanism of action and therefore to be suitable as therapeutic agents.

Chemically, EJP18 has unique characteristics: it bears three clusters of basic amino acids flanked by hydrophobic residues (LFM and double leucines at the N- and C-terminus, respectively) that might influence peptide–cell interaction. Even it is generally accepted that the first step for cellular entry of cationic CPPs is dependent on electrostatic interactions with polar headgroups of cell surface lipids, the presence of hydrophobic residues might enhance the capacity of this peptide to bind to the cell membrane and the efficiency for cargo delivery, similar to what the same authors have previously reported [12]. Further studies are needed to evaluate the critical portion(s) of the peptide. On the other hands, the mutual cooperation between positive and hydrophobic amino acids may promote the interaction of the peptide with specific membrane domains [13]. This could imply an increase in the specificity of CPPs in dependence on the specific lipid composition of different cell types. It would be an interesting test this hypothesis in model membranes in vitro whose composition can be quantitatively defined.

Overall, these data highlight that EJP18 represents a novel membrane-active peptide that could be used as a novel carrier for biomedical applications. However, this work also leaves some unresolved questions. One very intriguing issue regards whether EJP18 could interact with the EGF receptor at the plasma membrane. If yes, does EJP18 affect the EGFR trafficking and signalling? This could explain the inverse correlation between the expression levels of EGFR and EJP18 cytotoxicity that the authors observed. In the cells with high EGFR levels, the interaction between EJP18 and EGFR could be favoured and this, in turn, might lead to alter the peptide fate from one side, and the trafficking of EGFR itself further dysregulating its signalling from the other side. The understanding of these mechanisms is of fundamental importance for anticancer therapies since EGFR expression is quite variable in different cancer types and in various stages of tumour progression [14,15].

The observation by the authors that juxtamembrane regions of many receptor tyrosine kinases (such as ErbB2, ErbB3, FGFR and NTRK family, MET, etc.) are rich in positive charged residues and, therefore, might include potential active peptides is very intriguing, thus offering great potential for targeted cancer therapies.

Another interesting question is whether EJP18 is able to oligomerize in vitro and in vivo (in presence of cell membranes) and in dependence on its concentration. Certainly, a better comprehension of the biophysical properties of this peptide will shed light on the mechanisms of its uptake and in its intracellular delivery performance.

In conclusion, for their unique characteristics, CPPs represent powerful therapeutic agents. However, their use is still limited in clinical for some their drawbacks such as low cell/tissue specificity, low selectivity for intracellular targets, and low stability in the bloodstream.

In the near future, much effort should be dedicated for developing novel strategies to overcome these limitations. From one side, it will be important searching for new active peptides with better efficacy, as Eissa et al. did, or improving the performance of already existing CPPs (e.g. through chemical modifications). From the other side, it will be fundamental a profound understanding of the relationship between peptides and cellular components in terms of biophysical interactions and molecular dynamics. For instance, understanding how the lipid composition of the plasma membrane and how lipid domains at the cell surface could influence the entry, dynamics and fate of CPPs and how surface proteins might positively or negatively affect the CPPs’ behaviour remains an important and unresolved challenge. Many studies have shown that membrane organization is different and differently regulated in various cell types and can change in relation to the diverse metabolic state [16–18]. Next challenge is unravelling which are the differences between healthy and unhealthy cells (e.g. in terms of the surface lipid composition or the viscoelastic properties of the surface, for instance between cancer vs non-cancer cells, etc.), and this would help to generate more selective CPPs.

Finally, once entered, how could cytosolic and/or luminal environment influence the CPP properties and vice-versa how could CPPs affect the cellular components? Hence, all these critical aspects should be the subjects of future studies.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the POR Campania FESR 2014-2020 ‘SATIN' grant.

Contents

Figures & Tables

CPPs mediate different biological effects in dependence on their concentration. At higher concentrations, CPPs lead to membrane blebbings and protrusions, loss of cell viability through an unknown mechanism. At lower concentrations, CPPs penetrate into cell where they might exert their action in dependence on the diverse peptide and/or CPP-cargo conjugates (red question marks). The precise mechanism of endosomal CPPs’ escape from endosomes remains elusive.

CPPs mediate different biological effects in dependence on their concentration. At higher concentrations, CPPs lead to membrane blebbings and protrusions, loss of cell viability through an unknown mechanism. At lower concentrations, CPPs penetrate into cell where they might exert their action in dependence on the diverse peptide and/or CPP-cargo conjugates (red question marks). The precise mechanism of endosomal CPPs’ escape from endosomes remains elusive.